Conditions were determined for achieving the highest level of binding of metabolized B(a)P to double stranded salmon sperm DNA in the presence of microsomal metabolizing system. The binding level was up to 50 fold higher than that reported in the literature. Thus, an appropriate substrate DNA has been prepared for the experiments leading to the elucidation of the nature of the putative (BPase) activity. A rapid method of purification of 5, 6-dihydroxydihydrothymine (thymine glycol, TG) was developed. TG is used as a marker to determine the nature of modified thymine residues in DNA and to determine the specificity of enzymatic activity directed against such modified DNA, including benzo(a)pyrene modified DNA.